Pneumonia is a major public health burden. Klebsiella pneumoniae (K. pn) is a major cause of nosocomial and community-acquired pneumonia, particularly in immunocompromised or debilitated patients, and antibiotic- resistant strains have become an increasing public health concern. The alveolar macrophage (AM), the sentinel cell of lung innate immunity, is the first responder to infection. Surfactant protein A (SP-A), an innate immune protein, is secreted by the lung epithelium into alveoli where the AM reside. SP-A regulates many AM functions and has a major effect on the AM proteome, including changes in proteins related to the cytoskeleton. How proteins, such as cell surface proteins and those identified by our proteomics studies, interact to bring about specific AM functions is not known. We will address this knowledge gap using the Toponome Imaging System (TIS), the first microscopic system with high functional resolution (~40nm) that can localize up to 100 proteins in a single cell. Studies with TIS have shown how proteins are organized into clusters or combinatorial molecular phenotypes (CMP), which are functional units of protein signaling networks, and how the function of groups of CMPs depend on a few proteins, termed lead proteins. We hypothesize that SP-A-regulated AM functions +/- K. pn infection depend on specific CMPs, the integrity of which depend on lead proteins, and that both the CMPs and lead proteins differ under different conditions. Two Specific Aims are proposed: 1) To study and compare CMPs in AM from K. pn-infected wild type and SP-A -/- mice, identify CMPs unique to each study group, and identify the lead protein(s) in CMP groups, and 2) to inhibit the lead protein(s) of selected CMPs (from Aim 1) and investigate the impact of this inhibition on cytoskeletal changes, cell size, and disruption of CMP integrity. The proteins probed will include AM cell surface molecules (some known to interact with SP-A), SP-A-regulated proteins (with emphasis on cytoskeletal proteins), and proteins that distinguish macrophage subsets. Inhibition will be accomplished using blocking antibodies, or other approaches based on the nature of lead molecule(s) and reagent availability. Data analysis will identify CMP motifs that are specific for and distinguish among the study conditions. For the inhibition studies priority will be given t lead proteins identified in CMP motifs that include the largest number of individual CMPs. The knowledge gained will contribute to our understanding of how cell surface and intracellular proteins interact in the regulation of the cytoskeleton and identify molecules instrumental in these interactions. The successful completion of this will provide insight into lung pathophysiology in response to infection, and the role of the innate immune protein SP-A in this process. Importantly, identification of CMP and lead proteins associated with AM morphology and cytoskeletal changes in this proof of principle study, will provide the foundation for future studies where lead proteins responsible for a specific, more complex outcome, such as phagocytosis, could be identified and targeted for therapeutic intervention to enhance or inhibit AM function.